question about cycloheximide

theodorn at medlib.georgetown.edu theodorn at medlib.georgetown.edu
Thu Aug 27 09:18:03 EST 1998


In article <6s14ik$r8t at mserv1.dl.ac.uk>,
  Nico Dantuma <nico.dantuma at mtc.ki.se> wrote:
> Hi,
>
> I tried in an experiment to inhibit protein synthesis with cycloheximide.
> To my surprise I didn't see any effect of the cycloheximide incubation.
> However I am not completely sure abou this since it could also be due to my
> monitoring system (GFP fusion in FACS analysis). Since I have never used
> cycloheximide I did a simple experiment to test the activity of
> cycloheximide by incubating a confluent HeLa culture overnight with 100
> ug/ml cycloheximide. I expected a high mortality because of the
> cycloheximide but I didn't observe a clear difference with a control culture.
>
> 1) Can I conclude from the latter experiment that my cycloheximide is not
> inhibiting protein synthesis under the conditions that I used?
>
> 2) If so, what did I do wrong? I made a 10 mg/ml stock solution in PBS,
> filter sterilized it and diluted it 100 fold in the incubation medium. Do I
> have to add a supplement?
>
> Thanks in advance,
>
> Nico Dantuma
> ------------------------------------------
> Nico Dantuma
> Microbiology and Tumor Biology Center
> Karolinska Institute
> Box 280
> S-17177 Stockholm
> Sweden
> Phone: + 46 8 7286281
> Fax: +46 8 331399
>

It takes awhile for cells to die (or at least until the look dead) from the
inhibition of protein synthesis, as anyone who has used G418 selection can
tell you.  Overnight is not long enough, especially if the cells are
confluent since they seem to resist to effects of the drug better (maybe
because they have already shut down a lot of synthesis).  Your best bet is to
do a pulse-labelling with a radioactive amino acid and determine the
TCA-precipitable counts.  The conditions you describe should work well; in my
hands 100 ug/ml cycloheximide inhibited greater than 98% of TCA pptable
counts in HeLa cells (I make and use the cyclo essentially as you have
described it).

Nick Theodorakis

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